Application of siRNA in improving sensitivity of triple negative breast cancer cell line MDA-MB-468 to tamoxifen
By inhibiting the growth and clonal formation ability of TNBC cells against PRPF8, the sensitivity of triple-negative breast cancer cells to tamoxifen was improved, and the problem of lack of TNBC therapeutic targets was solved, which significantly affected cell growth and clonal formation. , effective inhibition of MDA-MB-468 cells was achieved.
Patent Information
- Application Number
- CN202411333413.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-24
- Publication Date
- 2024-12-27
AI Technical Summary
Triple-negative breast cancer (TNBC) treatment is difficult to effectively inhibit cell growth and clonal formation, resulting in high recurrence and metastasis rates. The existing targeted therapeutic targets are almost negative in TNBC, increasing the difficulty of treatment.
Using specific siRNA sequences (siRNA PRPF8-1 and siRNA PRPF8-2) to target mRNA precursor splicing factor 8 (PRPF8), improving the triple-negative breast cancer cell line MDA-MB-468 against tamoxifen by inhibiting its expression Sensitivity, which in turn affects cell growth and clonal formation ability.
It significantly reduced the growth rate and clonal formation ability of MDA-MB-468 cells, improved the sensitivity of cells to tamoxifen, had strong targeting siRNA, high knockdown efficiency, and significant expression inhibition effect.
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Figure CN119193588A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of breast cancer, and particularly relates to the application of siRNA in improving the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen. Background Art
[0002] Breast cancer can be divided into four types according to molecular classification: Luminal A breast cancer, Luminal B breast cancer, HER-2 positive breast cancer and triple negative breast cancer. Among them, triple negative breast cancer (TNBC) has malignant characteristics such as easy metastasis, high recurrence rate and low age of onset, which has become a difficulty in clinical treatment. At present, the clinical treatment options for TNBC mainly include surgical treatment, chemoradiotherapy and targeted therapy. Unfortunately, the postoperative recurrence rate of TNBC is as high as 25% in clinical practice, and within five years after receiving chemoradiotherapy, the number of patients with recurrence or distant metastasis is as high as 50%. In addition, the conventional targets for clinical treatment of breast cancer are almost all negative in TNBC, which greatly increases the difficulty of treatment. Therefore, it is of great significance to develop new effective therapeutic targets to improve the survival rate of TNBC patients.
[0003] Pre-mRNA processing factor 8 (PRPF8) is a key splicing factor in the alternative splicing process. It can interact with multiple factors in the spliceosome complex and form the catalytic center of the spliceosome complex. It is crucial to maintain the accuracy and efficiency of the entire splicing process. PRPF8 has been studied in depth as a key factor in retinitis pigmentosa. Recent studies have found that PRPF8 is closely related to cancer, and its mutation or abnormal expression may produce splicing isoforms that are more favorable to tumor cell growth and metastasis. However, so far, the key targets and regulatory mechanisms of PRPF8 in regulating the growth of TNBC have not been reported. Summary of the invention
[0004] The present invention provides the use of siRNA in improving the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen. The PRPF8 expression inhibitor can inhibit the growth of triple-negative breast cancer cells and inhibit cell clone formation, and can be used for clinical treatment of triple-negative breast cancer.
[0005] The application of siRNA of the present invention, wherein the siRNA is a double-stranded RNA molecule consisting of a sense strand and an antisense strand, including siRNA PRPF8-1 and siRNA PRPF8-2;
[0006] The nucleotide sequence of the sense strand of the siRNA PRPF8-1 is shown in SEQ ID No.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.2; the nucleotide sequence of the sense strand of the siRNA PRPF8-2 is shown in SEQ ID No.3; and the nucleotide sequence of the antisense strand is shown in SEQ ID No.4;
[0007] The siRNA is used to improve the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen.
[0008] The nucleotide sequence of the positive strand of the siRNA PRPF8-1 is GTACCATACTCCCAATGTTdTdT.
[0009] The antisense strand nucleotide sequence of the siRNA PRPF8-1 is AACATTGGGAGTATGGTACdTdT.
[0010] The nucleotide sequence of the positive strand of the siRNA PRPF8-2 is GCATGCGCCTCATGAAACAdTdT.
[0011] The antisense strand nucleotide sequence of the siRNA PRPF8-2 is TGTTTCATGAGGCGCATGCdTdT.
[0012] Furthermore, the improvement refers to increasing the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen.
[0013] Furthermore, siRNA improves the tamoxifen sensitivity of the triple-negative breast cancer cell line MDA-MB-468 cells by inhibiting the growth and cloning ability of the triple-negative breast cancer cell line MDA-MB-468 cells.
[0014] A pharmaceutical composition of the present invention comprises the siRNA for inhibiting PRPF8 expression according to claim 1.
[0015] The present invention has the following beneficial effects:
[0016] 1) The siRNA of the present invention is specific to PRPF8 and has strong targeting. 2) The present invention significantly affects the growth and clone formation ability of the triple-negative breast cancer cell line MDA-MB-468 cells, and affects the tamoxifen sensitivity of MDA-MB-468 cells. After transfection with siRNA, the sensitivity of MDA-MB-468 cells to tamoxifen is significantly enhanced. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1Figure 2 shows the growth and clone formation ability of breast cancer cell MDA-MB-468 after knocking down PRPF8; (a): The efficiency of knocking down PRPF8 by two groups of siRNA in MDA-MB-468 cells. (b): Cell proliferation curve after PRPF8 knockdown; (c): Clone formation experiment after PRPF8 knockdown. (d): The number of cell colonies formed by target cell clones after PRPF8 knockdown. DETAILED DESCRIPTION
[0018] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.
[0019] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.
[0020] Example 1
[0021] Cell culture: MDA-MB-468 cell line was obtained from Shanghai Cell Bank of Chinese Academy of Sciences. Cell culture was performed in L15 medium supplemented with 10% calf serum in an air incubator at 37°C.
[0022] siRNA transfection: Take MDA-MB-468 cells and count them for plating. The number of cells in a 6-well plate is 5×10 5 / well cells, and 24 hours later, siRNA PRPF8-1 and PRPF8-2 were transfected into triple-negative breast cancer cells MDA-MB-468, respectively. The cells were cultured in a 37-degree air incubator.
[0023] Cell growth change detection:
[0024] After PRPF8 knockdown for 48 h, the cells were counted and plated. The number of cells in a 96-well plate was 1×10 4 Each group was cultured for 1, 3, and 5 days, and then a mixed solution of 10 μL CCK-8 and 100 μL culture medium was added to each well in the dark. After being placed in the incubator for 1 hour, the absorbance of each well at 450 nm was detected using an ELISA reader. The effect of PRPF8 knockdown on the proliferation ability of TNBC cells was analyzed.
[0025] Cell clone formation ability test
[0026] After PRPF8 knockdown for 48 h, the cells were counted and plated, and 8×10 2 / well, and repeatedly and carefully blow and beat several times to disperse the cells as much as possible. Set up 3 sets of duplicate wells and place them in an incubator at 37°C for constant temperature culture. Change the culture medium every three days. Observe the growth of cell clusters under a microscope until the cell clusters grow to a suitable size (the clusters can be clearly seen with the naked eye), fix and stain the cells in methanol, stain them with crystal violet, and then count the cell clusters in each well.
[0027] Experimental Results
[0028] After specific siRNA was transfected into MDA-MB-468 cells and cultured for 48 hours, the expression level of PRPF8 gene was detected. The results showed (as shown below) Figure 1 A), the expression of PRPF8 mRNA in the knockdown groups 8-1 and 8-2 was significantly lower than that in the control group NC (knockdown efficiency>70%, p<0.01). After knocking down PRPF8 in MDA-MB-468 cells for 48 hours, CCK-8 was used to detect changes in cell growth. The results showed that after PRPF8 knockdown, the growth rate of the TNBC cell MDA-MB-468 cell line was significantly reduced, and the cell clone formation ability was also significantly reduced. The above results all indicate that there is an important connection between PRPF8 and TNBC growth (see Appendix Figure 1 ).
Claims
1. Application of siRNA, characterized in that The siRNA is a double-stranded RNA molecule consisting of a sense strand and an antisense strand, including siRNA PRPF8-1 and siRNA PRPF8-2; The nucleotide sequence of the sense strand of the siRNA PRPF8-1 is shown in SEQ ID No.1, and the nucleotide sequence of the antisense strand is shown in SEQ ID No.2; the nucleotide sequence of the sense strand of the siRNA PRPF8-2 is shown in SEQ ID No.3; and the nucleotide sequence of the antisense strand is shown in SEQ ID No.4; The siRNA is used to improve the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen.
2. The use of siRNA according to claim 1, characterized in that The improvement refers to increasing the sensitivity of triple-negative breast cancer cell line MDA-MB-468 cells to tamoxifen.
3. The use of siRNA according to claim 1, characterized in that The siRNA improves the tamoxifen sensitivity of the triple-negative breast cancer cell line MDA-MB-468 cells by inhibiting the growth and cloning ability of the triple-negative breast cancer cell line MDA-MB-468 cells.
4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the siRNA for inhibiting PRPF8 expression according to claim 1.